In situ heat induced antigen recovery and staining apparatus and method

ABSTRACT

An automated in situ heat induced antigen recovery and staining method and apparatus for treating a plurality of microscope slides. The process of heat induced antigen recovery and the process of staining the biological sample on the microscope slide are conducted in the same apparatus, wherein the microscope slides do not need to be physically removed from one apparatus to another. Each treatment step occurs within the same reaction compartment. The reaction conditions of each reaction compartment for treating a slide can preferably be controlled independently, including the individualized application of reagents to each slide and the individualized treatment of each slide. The reagents are preferably held in a reagent dispensing strip similar to a “blister pack”.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 10/388,710,filed Mar. 14, 2003, which is a continuation of U.S. Ser. No.09/612,605, filed Jul. 7, 2000, now U.S. Pat. No. 6,534,008, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/142,789,filed Jul. 8, 1999, each of which is hereby incorporated by referenceherein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

BACKGROUND

The present invention is related to the field of treating samples onmicroscope slides and more specifically to the field of heat inducedantigen recovery and staining.

Antigen recovery, also known as antigen unmasking, antigen epitopeunmasking, antigen retrieval or heat induced epitope recovery (HIER) isa process in which biological samples (e.g., cells, tissues, blood,fluids) are treated under heat with a series of aqueous or non-aqueousreagents and buffers (e.g., citrate, EDTA, and urea) for the purpose ofexposing the presence of specific types of antigens or biochemicalfeatures in the biological samples. HIER is regarded as a pre-treatmentprocedure to be performed prior to the beginning of a specific stainingprotocol to identify cellular components.

Biological samples must be preserved after removal from the body. Thispreservation process, known as fixation, kills and localizes thebiological material. One of the most common fixatives used widely in thepreservation of biological materials is formalin, a 10% aqueous solutionof formaldehyde. This fixative, along with other widely utilizedfixatives, produces a cross-linking network around specific sites in thebiological material. These sites are known as antigens, and during thefixation process become “masked,” by the fixative and thus “invisible”to detection by certain stains. HIER is used as a pre-treatment processto “unmask,” “retrieve” or “recover.” This process is usually conductedon formalin fixed paraffin embedded tissue sections or cellularpreparations mounted on microscope slides.

U.S. Pat. No. 5,244,787 teaches a process of antigen retrieval whereinone or more slides are placed in an aqueous solution within a microwaveoven and heated to boiling or near-boiling temperatures. These slidesare all treated together in a rack that has been placed in a bath of thesolution. The slides are near boiling temperatures for 5-30 minutes,generally around 10 minutes. Due to excessive evaporation from the bath,the patent teaches that the solution should not drop below thebiological sample on the slide because drying out of the sample isdeleterious. This process further teaches that after boiling ornear-boiling for several minutes, usually 5 minutes, one may have to addmore solution to the container to prevent the solution from excessiveevaporation and subsequent exposure of the samples on the slides. Afterthe addition of more liquid, the process is continued until the desiredtime is completed. The disclosure of U.S. Pat. No. 5,244,787 is limitedto the use of a microwave oven as the source of heating. More recentadvances, which have been published, include the use of different typesof heating devices such as electric pressure cookers, electric steamers,electric conduction heating surfaces utilizing pressure cookers,steamers, and also steam driving autoclaves (J. of Pathology,179:347-352, 1996; Biotechnic & Histochemistry, 71(5):263-270, 1996;Biotechnic & Histochemistry, 71(4): 190-195, 1996; J. of Histochemistry& Cytochemistry, 45(3): 327-342, 1997).

Although these published methodologies treat the biological sample withdifferent types of solutions and with varying types of chemicals and atdifferent pH's, all teach that all slides are treated together in a bathof the heated solution. After the slides have cooled for a period oftime, they are removed from the heating device and they are transferredto another apparatus where they are manually or automatically stainedusing various reagents. This pre-treatment process of heating andremoving the slides from the heating device for staining in a separatedapparatus is highly cumbersome and inefficient. The only automated HIERor antigen retrieval instrument available is the BIOGENEX i1000. Thisinstrument, however, still employs the use of the known technology oftreating the slides as a group in a container filled with heatedsolutions. A technician must still remove the slides from the antigenretrieval (heating) instrument and place them in an automated stainerinstrument to complete the required staining protocol.

As noted herein, no currently available automated or semi-automatedstaining instruments specifically teach the ability to heat an aqueousor non-aqueous liquid for the unmasking of antigens. The instrumentsthat do automated or semi-automated staining limit their scope to thattask alone, and don't address the task of HIER or antigen retrievalpre-treatments. U.S. Pat. Nos. 5,073,504 and 4,847,208 teach use of achamber for enclosing and staining a microscope slide but neitherteaches use of a heating device to boil a liquid and the user must addthe primary antibody manually through a hinged door on top of thechamber. U.S. Pat. Nos. 4,777,020; 4,798,706; and 4,801,431 teach use ofa vertical staining “capillary gap” methodology wherein two specialslides placed front to front causing an air gap through which liquidsare drawn by capillary movement. This gap can only hold a small volume(approx. 300 microliters) of liquid. If heated to near boilingconditions the liquid would evaporate through all four open sides,immediately causing the biological sample to dry. This end result istrue also for another capillary gap instrument, shown in U.S. Pat. No.5,804,141. U.S. Pat. Nos. 5,595,707; 5,654,200; 5,654,199, 5,595,707;and 5,650,327 teach reducing evaporative loss by utilizing an oil layeron top of the aqueous layer. This is somewhat effective in reducing theamount of evaporative loss at 37° C. but the volume of the aqueous layer(approx. 300 microliters) is again minimal, and if heated to boiling,would cause the aqueous layer to dry out leaving only the oil layerpresent thus damaging the biological sample unless more aqueous reagentwas applied during the treatment process. U.S. Pat. No. 5,425,918 alsoteaches use of small amounts of liquids that are sprayed on the slideand can only heat the slide to 37° C. U.S. Pat. Nos. 5,645,144 and5,947,167 teach use of an open top chamber present around the slide anduse a rotating cover above the slides to reduce evaporation. There is noteaching of high temperature heating of a liquid for a substantialamount of time. Further, even if one would increase the temperature ofthe slide, the loosely rotating top of the chamber would allow so muchevaporative loss that the solution would never reach boiling or nearboiling temperatures, nor would it maintain the boiling conditions for10 minutes or longer. U.S. Pat. No. 5,645,114 teaches use of smallvolumes of liquids (up to 500 microliters) and has no ability to stopevaporative loss if the slide temperature reaches boiling conditions.

As a result, none of these systems could hold sufficient liquid on topof a slide (e.g., 4 ml) and are enclosed in a chamber which is properlyvented to minimize the energy loss from evaporation to cause sufficientheating to boil the liquid on the slide for the length of time generallyrequired to cause antigen unmasking (e.g., 10-30 minutes).

There remains a need for an apparatus which can perform the task of HIERwith subsequent staining treatment without the need of switching theslides from one apparatus to another and wherein the treatment of allmicroscope slides can occur simultaneously thereby increasingefficiency. Of the automated stainers available today, there is not oneinstrument that has the ability to overcome the inherent problems ofheating an aqueous or non-aqueous solution at a sufficient volumewithout the undesirable effect of evaporative heat loss and subsequentvolume decrease of the solution. The negative effects of evaporation aresignificant. The ability of a liquid to reach boiling or near boilingtemperature on a microscope slide is dependent on the containment andcontrol of the steam or vapor generated during the heating process. Itis the object of the invention contemplated herein to provide acompletely automated HIER apparatus which can recover antigens withmultiple types of recovery buffers simultaneously, each specific to itsrespective microscope slide and which can also be used to stain themicroscope slides as well.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an apparatus of the invention (shownwithout a pressing element for crushing a reagent capsule).

FIG. 2 is a cross-sectional view of the apparatus of FIG. 1 (shown witha pressing element for crushing a reagent capsule).

FIG. 3A is a cross-sectional view of the apparatus of FIG. 1 (shown witha reaction compartment having a raised slide support surface) takenthrough line 3A-3A.

FIG. 3B is a cross-sectional view of the apparatus of FIG. 1 (shown witha reaction compartment having a lowered slide support surface) takenthrough line 3B-3B.

FIG. 4 is a cross-sectional view of an alternative embodiment of theapparatus of the present invention having an alternate type of slidesupport surface.

FIG. 5 is a perspective view of a reagent strip of the presentinvention.

FIG. 6 is a cross-sectional view of the reagent strip of FIG. 5 takenthrough the line 6-6.

FIG. 7 is an elevational view of a modular apparatus containing aplurality of the apparatus of FIG. 1.

FIG. 8 is a flow chart showing a preferred sequence of steps in themethod of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to an automated method and apparatusfor treating biological samples on microscope slides for unmasking(“retrieving” or “recovering”) epitopes or antigens of the biologicalsamples and then staining or otherwise treating the biological samples.The automated apparatus comprises an array of individual reactioncompartments, each of which is used to treat a single microscope slide(also referred to herein as a “slide”), wherein each reactioncompartment preferably can function and can be controlled independentlyof the other reaction compartments in the array. Each reactioncompartment in the array comprises a support element comprising asurface upon which a microscope slide can be supported and positionedadjacent or inserted into the compartment for treatment with a reagent.The support element further comprises, in a preferred embodiment, aconduction type heating element for heating the microscope slide to apredetermined treatment temperature when desired. The support elementwith the microscope slide thereon can be raised into or adjacent thereaction compartment for treatment of the microscope slide, or loweredor removed from the reaction compartment for placement of a microscopeslide onto or removed from the support surface or for removal of areagent or rinsing solution from the microscope slide during thetreatment process.

Reagents, such as antibodies, enzymes, rinse buffers, antigen recoverybuffers, or stains, are contained in an individualized reagentdispensing strip which is specific for each microscope slide to betreated. Since each microscope slide and reaction compartment isgenerally provided with its own reagent dispensing strip, eachmicroscope slide can be treated independently with a different set ofreagents (a particular treatment protocol) while being treatedsimultaneously with other microscope slides. Similarly, in an especiallypreferred embodiment of the invention, each microscope slide can beheated separately, as well as treated with a different treatmentprotocol.

The apparatus of the present invention therefore comprises, in apreferred embodiment, a plurality of individualized reactioncompartments in a chamber which can be substantially closed forminimizing evaporation during heating. A microscope slide can besupported in each reaction compartment, and each microscope slide can beheated separately therein. A reagent dispensing strip containing aplurality of individually contained reagents (reagent “bubbles”,“blisters” or “capsules”) is positioned upon an upper portion of eachreaction compartment, and at an appropriate time, a reagent from eachreagent dispensing strip is expelled from a reagent capsule undercompression and is thereby applied to the biological sample on themicroscope slide. Or, a reagent, such as an antigen recovery buffer canbe introduced via a separate dispenser. The term “reagent” is definedherein to include any type of fluid material that may be applied to thebiological material on the microscope slide, including antibodies,stains, enzymes, buffers, rinses, or washes, or any other materialapplied in the process of antigen recovery or treating the biologicalmaterial on the microscope slide to be viewed under the microscope.

During an antigen recovery step, the microscope slide, sample, andantigen recovery buffer thereon are heated to an appropriate temperaturefor a predetermined duration to cause the antigen recovery buffer toreact with the sample on the microscope slide, after which the antigenrecovery buffer is removed from the microscope slide, preferably bywashing or flooding the microscope slide or chamber containing themicroscope slide with a rinse buffer and allowing the rinse buffer todrain off by gravity or by blowing the solution off the microscope slideusing pressurized air. Each microscope slide may be treated in the samemanner, or may be treated with different reagents using a differenttreatment protocol, preferably simultaneously, yet independently.

When a reagent is provided via a reagent dispensing strip, the apparatusis preferably equipped with a drive mechanism for causing the reagentdispensing strip to be advanced in a forward direction wherein eachreagent capsule in succession is positioned above an aperture in thecompartment through which the reagent in the capsule is delivered. Thereagent dispensing strip may be advanced using rollers positioned alongthe upper end of the compartment or a pushing mechanism which pushesupon the rear end of the reagent dispensing strip. The reagent in thereagent capsule of the reagent dispensing strip is to be applied to themicroscope slide by a pressing mechanism which, in a preferred version,compresses and thereby crushes the reagent capsule and causes thereagent to be expelled and deposited directly onto the microscope slide.

In a preferred method of the present invention, a plurality ofmicroscope slides, each having thereon a sample to be treated, isprovided. Each microscope slide is positioned upon a support elementwhich is then moved into an application position. A plurality of reagentdispensing strips is provided, one for each microscope slide to betreated. Each microscope slide is subjected to an antigen recovery stepthen is treated by applying a reagent from its corresponding reagentdispensing strip. Each microscope slide can be handled differently, ifdesired, during the treatment cycle. After a predetermined duration, themicroscope slide and support element is moved to a removal positionwherein the reagent is removed, preferably in between reagentapplications, by treatment with a rinsing solution to remove the reagentprior to further treatment. Each microscope slide can be treatedaccording to the treatment protocol specific to that sample or thatparticular microscope slide. All microscope slides may be treated usingthe same protocol, or one or more, or all, of the microscope slides maybe treated using a different protocol.

An example of a treatment protocol comprises:

-   -   1) antigen recovery, 10 minutes at 98° C.,    -   2) cool, 20 minutes,    -   3) rinse buffer,    -   4) primary antibody, 30 minutes,    -   5) rinse,    -   6) biotinylated linking antibody, 10 minutes,    -   7) rinse buffer,    -   8) peroxidase labeled streptavidin label,    -   9) rinse buffer,    -   10) 3,3′-diaminobenzidine chromogen,    -   11) rinse buffer,    -   12) chromogen enhancer,    -   13) rinse buffer, and    -   14) counter stain.

A variety of other treatment protocols are well known to those ofordinary skill in the art and further discussion of them herein is notdeemed necessary. Each microscope slide, if necessary, may be heatedprior to application of the reagent, if necessary, then may be cooled asthe reagent is removed, then reheated, if necessary, prior to or afteraddition of the next reagent. The entire process is run automaticallyonce the microscope slide is disposed onto the support element, and thereagent dispensing strip is positioned upon the upper side of thereaction compartment.

Turning now to the drawings, a specific embodiment of the apparatus ofthe present invention is shown in FIGS. 1-6. Although FIGS. 1-6 show apreferred version of the invention, it will be understood that theembodiment shown in FIGS. 1-6 is but one of many possible versions ofthe apparatus enabled herein which will come to the mind of a person ofordinary skill in the art.

Shown in FIG. 1, and designated therein by the general reference numeral10 is an antigen recovery and staining apparatus constructed inaccordance with the present invention. The antigen recovery and stainingapparatus 10 comprises a treatment chamber 12 which further comprises aplurality of reaction compartments 14 (see FIGS. 2-4). Preferably thetreatment chamber 12 generally comprises from 10 to 20 reactioncompartments 14 but may contain more or fewer. Each reaction compartment14, when enclosed, minimizes evaporation of a reagent solution when amicroscope slide is exposed to high temperature pretreatment conditions.Each reaction compartment 14 has an upper side 16 having an opening 18therein, a lower side 20, and a pair of sidewalls 22 which extend fromthe rear end 23 a of the treatment chamber 12 to the front end 23 b ofthe treatment chamber 12. Positioned above each reaction compartment 14is a reagent dispensing strip holder 24 for holding and guiding areagent dispensing strip 26 (see FIGS. 5 and 6). Each reagent dispensingstrip 26 has a front end 28 and a rear end 30 and a plurality ofcapsules 32 made of a crushable plastic material such as polyethylene oranother suitable material (e.g., polypropylene or polystyrene) and whichmay include one or more multiple capsules 32 a. The size of each capsule32 or multiple capsule 32 a may be adjusted to accommodate the amount ofreagent which is desired to be applied to a microscope slide 44. Eachcapsule 32 or multiple capsule 32 a contains a reagent or treatmentsolution which is intended to be applied to a biological material on themicroscope slide 44. Multiple capsule 32 a is useful in a method whereintwo or more reagents must be contained separately before being appliedto the microscope slide 44. When the multiple capsule 32 a is crushed bythe pressing mechanism 36, two or more reagents contained within thecapsule 32 a are combined and simultaneously applied to the microscopeslide 44.

Other embodiments of the reagent dispensing strip 26 and the reagentcapsule 32 and multiple capsule 32 a will readily be apparent to one ofordinarily skill in the art. For example, each reagent dispensing strip26 may comprise a one or more “blank” spaces for insertion ofindividualized capsules 32 by a user. Below each capsule 32 or multiplecapsule 32 a is an aperture or weak area 34 in the reagent dispensingstrip 26 through which the reagent in the capsule 32 or multiplecapsules 32 can be forced by a pressing mechanism 36. The “blank” spaceor space left by the puncturing of a capsule 32 or 32 a, or vents in thereagent dispensing strip 26 may function to release pressure, steam orvapors produced during the treatment process. The reagent dispensingstrip 26 is advanced in a direction 37 toward the front end 23 b of thetreatment chamber 12 by a reagent strip drive mechanism 38 driven, forexample, by an electric motor which in FIGS. 1, 3A and 3B is shown as apushing mechanism comprising a threaded shaft, but which may instead bya mechanism (not shown) comprising rollers which drive, draw or “pull”the reagent strip holder 24 in a forward direction 37.

Each reaction compartment 14 further comprises at its lower side 20 aslide support element 40 having a slide tray 42 upon which themicroscope slide 44 can be positioned and held for treatment. The slidesupport elements 40 together comprise a slide support assembly 39. Withthe microscope slide 44 disposed on the slide support element 40, theslide support element 40 and the microscope slide 44 are positioned inan application position to fit adjacent the lower side 20 of thereaction compartment 14, thereby constituting an openable bottom of thereaction compartment 14. The slide support element 40 further has aheating element 46 incorporated therein for heating the microscope slide44 as discussed elsewhere herein. In one embodiment, the slide supportelement 40 has a hinge 48 for enabling the slide support element 40 tobe moved (raised) into an application position (FIG. 3A) and therefromlowered (e.g., tilted) into an opened position (see FIG. 3B).Alternatively, the slide support element 40 may be raised and loweredinto position by another mechanism, such as a stepper motor 58 and screwdrive 59 mechanism (FIG. 4). Each reaction compartment 14 furthercomprises a manifold 50 which comprises, in a preferred embodiment, aplurality of reagent dispensing ports or elements including, for examplebut not limited to, an antigen recovery buffer dispenser 51 connectedvia an antigen recovery buffer supply line 51 a to an antigen recoverybuffer supply (not shown), a rinse buffer dispenser 52 connected via arinse buffer supply line 52 a to a rinse buffer supply (not shown) andan air pressure nozzle (pressurized air nozzle) 54 connected via an airline 54 a to an air supply (not shown). The antigen recovery bufferdispenser 51 applies an antigen recovery buffer to the microscope slide44 for the antigen recovery treatment step prior to staining or otherpreparation of the biological material on the microscope slide 44. Therinse buffer dispenser 52 applies a rinse buffer 56 to the microscopeslide 44 to rinse a reagent from the microscope slide 44. The airpressure nozzle (pressurized air nozzle) 54 functions to clear away arinse buffer 56 from the microscope slide 44. Dispensers 51 and 52 maybe used to dispense other reagents, and may constitute more than, orfewer than, the dispensers shown in FIGS. 2, 3A, 3B, and 4. Themicroscope slide 44 is generally disposed in a removal position forfacilitating removal of the rinse buffer 56 as shown in FIGS. 1 and 3B.Each slide support element 40, in a preferred embodiment, can be heatedor moved independently of any other slide support element 40, althoughone of ordinary skill in the art can envision that the slide supportelements 40 may be designed to operate in concert, i.e., simultaneously.Each reaction compartment 14 preferably can contain a volume of up to 15ml.

The antigen recovery and staining apparatus 10 can be controlledautomatically wherein predetermined sequences and operations are carriedout using various electromechanical systems which are not shown butwhich are well known to those of ordinary skill in the art. For example,each of the steps of raising into a treatment position and lowering intoa removal position each of the slide support elements 40, applying anantigen recovery buffer, advancing each reagent dispensing strip 26,compressing each capsule 32 or 32 a of the reagent dispensing strip 26,heating each microscope slide 44 on the slide support surface 40,applying a rinse buffer 56 to the microscope slide 44, removing therinse buffer 56 or other reagent from the microscope slide 44, andtreating each microscope slide 44 independently can be automaticallycontrolled and programed using programming methods and devices wellknown in the art. Because each reaction compartment 14 and slide supportelement 40 can be controlled independently, a microscope slide 44 caneven be removed or inserted even while other reaction compartments 14are in operation.

Preferably, a microprocessor, not shown, controls the antigen recoveryand staining apparatus 10. That is, an operator programs themicroprocessor with information such as which reaction compartments 14are to be used and to what temperature each is to be heated and at whichsteps, then programs the particular treatment protocol to be performedon the sample on each microscope slide 44 on each slide support element40. Variables in these protocols can include the particular type ofreagent dispensing strip 26 to be used, the time that each reagent ortreatment solution on the reagent dispensing strip 26 will be allowed toreact with the sample on the microscope slide 44, whether the microscopeslide 44 will be heated, and if so to what temperature and for how long,and the manner in which the microscope slide 44 will be rinsed, forexample. Other variables not listed herein may also be programmed.

The invention may further comprise a modular apparatus 60 comprising aplurality of antigen recovery and staining apparatuses 10 each servingas an individual module in the modular apparatus 60. The individualmodules can be “stacked” together for example, as shown in FIG. 7, ormay be oriented in any other desirable manner.

Shown in FIG. 8 is a schematic drawing which describes the preferredmethod of the present invention. In the first step, a microscope slide44 which has a sample disposed thereon is provided, and is disposed ontoa slide support element 40 which is moved into an application ortreatment position adjacent or against the reaction compartment 14. If aplurality of microscope slides 44 are supplied, each microscope slide 44is disposed on a separate microscope slide support element 40 and themicroscope slides 44 are moved independently or simultaneously into anapplication position.

Once in the application position, an antigen recovery buffer isinitially applied to the sample on the microscope slide 44. Microscopeslide 44 is then heated to a desired, predetermined temperature, forexample from about 140° C. to about 160° C. whereby the antigen recoverybuffer is heated to a temperature of from about 90° C. to 100° C., forexample. The microscope slide 44 is allowed to react with the reagentfor a predetermined length of time, for example, 10 to 30 minutes,preferably at 95°-98° C. Venting of steam may occur through small holes(not shown) in the reagent strip 26 or elsewhere in the reactioncompartment 14. Venting may occur through a vapor pressure releasedevice in the reaction compartment 14. It is not necessary to addadditional antigen recovery buffer during this step. After the reactionperiod is over, the slide support element 40 and the microscope slide 44thereon are moved (lowered or dropped) to a removal position, ifnecessary, where the antigen recovery buffer is removed from themicroscope slide 44, for example, by applying a rinsing solution orbuffer to the microscope slide 44 or by gravity or by pressurized air. Arinse solution or buffer may be applied and removed more than once fortreatment or for removal of a particular reagent before or afterlowering the microscope slide 44 to the removal position. It may bedesirable to add rinse buffer to the microscope slide 44 to cool themicroscope slide 44 prior to lowering the microscope slide 44 to theremoval position, for example, by adding rinse buffer 56 to the antigenrecovery buffer before the microscope slide 44 is moved to the removalposition. After the microscope slide 44 has been treated for antigenrecovery, another reagent can then be applied for treatment of thesample on the microscope slide 44. In this step, the microscope slide 44and slide support element 40 are then returned to the applicationposition, a reagent is applied, and is then removed after the treatmentperiod. The series of steps may be repeated. When the treatment of thesample is completed, the microscope slide 44 is removed from the slidesupport element 40 for further treatment or analysis apart from theantigen recovery and staining apparatus 10.

Changes may be made in the construction and the operation of the variouscomponents, elements and assemblies described herein or in the steps orthe sequence of steps of the methods described herein without departingfrom the scope of the invention as defined in the following claims.

1. An in situ antigen recovery and staining apparatus for treatingbiological samples on microscope slides, comprising: a slide supportassembly comprising a plurality of slide support elements, each slidesupport element able to support a microscope slide; a heating elementfor heating each microscope slide on each slide support element; and atreatment chamber comprising a plurality of closeable reactioncompartments, wherein each reaction compartment is automaticallyopenable for removal or application of reagents disposed on eachmicroscope slide on the slide support element.
 2. The apparatus of claim1 comprising at least one of: a dispenser for dispensing a quantity of arinse solution or reagent onto the microscope slide, and/or, a nozzlefor delivering a stream of pressurized air onto the microscope slide,and/or, a port for dispensing an antigen recovery buffer into thereaction compartment, and wherein each dispenser, port, and/or nozzlecan operate independently, and/or a portion of the reaction compartmentwhich is movable upwardly and downwardly, and/or, a portion of thereaction compartment which can be moved horizontally or tilted, and/or,a microprocessor for controlling a component of the apparatus.
 3. Areagent dispensing strip comprising at least one reagent capsule, andsized to be positioned on or inserted into a reagent dispensing stripholder of the apparatus of claim
 1. 4. An in situ antigen recovery andstaining apparatus for treating biological samples on microscope slides,comprising: a slide support assembly comprising a plurality of slidesupport elements, each slide support element able to support amicroscope slide; a heating element incorporated into or adjacent eachslide support element for independently heating each microscope slide oneach slide support element; and a treatment chamber comprising aplurality of closeable reaction compartments, wherein each reactioncompartment is automatically openable for removal or application ofreagents disposed on each microscope slide on the slide support element.5. The apparatus of claim 4 comprising at least one of: a dispenser fordispensing a quantity of a rinse solution or reagent onto the microscopeslide, and/or, a nozzle for delivering a stream of pressurized air ontothe microscope slide, and/or, a port for dispensing an antigen recoverybuffer into the reaction compartment, and wherein each dispenser, port,and/or nozzle can operate independently, and/or a portion of thereaction compartment which is movable upwardly and downwardly, and/or, aportion of the reaction compartment which can be moved horizontally ortilted, and/or, a microprocessor for controlling a component of theapparatus.
 6. A reagent dispensing strip comprising at least one reagentcapsule, and sized to be positioned on or inserted into a reagentdispensing strip holder of the apparatus of claim
 4. 7. An in situantigen recovery and staining apparatus for treating biological sampleson microscope slides, comprising: a slide support assembly comprising aplurality of slide support elements, each slide support element able tosupport a microscope slide, and wherein each slide support element isindependently movable; and a heating element incorporated into oradjacent each slide support element able to independently heat amicroscope slide on the slide support element.
 8. The apparatus of claim7 comprising a treatment chamber comprising a plurality of closeablereaction compartments wherein a reagent can be delivered independentlyor simultaneously into an inner space thereof or onto the microscopeslide supported on the slide support element, and/or, a microprocessorfor controlling a component of the apparatus, and/or, wherein eachreaction compartment has a volume of up to 15 ml.
 9. A reagentdispensing strip comprising at least one reagent capsule, and sized tobe positioned on or inserted into a reagent dispensing strip holder ofthe apparatus of claim
 7. 10. An in situ antigen recovery and stainingapparatus for treating biological samples on microscope slides,comprising: a slide support assembly comprising a plurality of slidesupport elements, each slide support element able to support amicroscope slide in a horizontal position, wherein each slide supportelement is independently movable; and a heating element incorporatedinto or adjacent each slide support element able to independently heat amicroscope slide on the slide support element.
 11. The apparatus ofclaim 10 comprising a treatment chamber comprising a plurality ofcloseable reaction compartments wherein a reagent can be deliveredindependently or simultaneously into an inner space thereof or onto themicroscope slide supported on the slide support element, and/or, amicroprocessor for controlling a component of the apparatus, and/or,wherein each reaction compartment has a volume of up to 15 ml.
 12. Areagent dispensing strip comprising at least one reagent capsule, andsized to be positioned on or inserted into a reagent dispensing stripholder of the apparatus of claim
 10. 13. An in situ antigen recovery andstaining apparatus, comprising: an assembly comprising a plurality ofencloseable reaction compartments wherein the operation of each reactioncompartment is controllable independently of each other reactioncompartment in the assembly, and wherein each reaction compartmentcomprises a movable slide support element for supporting a microscopeslide, each movable slide support element having a heating elementincorporated therein or adjacent thereto, and wherein each microscopeslide when disposed in each reaction compartment can be heatedindependently and treated independently of each other microscope slidein each other reaction compartment.
 14. The apparatus of claim 13wherein each microscope slide can be treated with a different treatmentprotocol simultaneously, and/or each encloseable reaction compartmentcan be vented by a vapor pressure release device to allow release ofsteam, vapor or pressure therefrom when the microscope slide is heated,and/or each encloseable reaction compartment is individuallyencloseable, and/or comprising a microprocessor for controlling acomponent of the apparatus.
 15. A reagent dispensing strip comprising atleast one reagent capsule, and sized to be positioned on or insertedinto a reagent dispensing strip holder of the apparatus of claim
 13. 16.An in situ antigen recovery and staining apparatus, comprising: anassembly comprising a plurality of encloseable reaction compartmentswherein the operation of each reaction compartment is controllableindependently of each other reaction compartment in the assembly, andwherein each reaction compartment comprises an individually movableslide support element for supporting a microscope slide, eachindividually movable slide support element having a heating elementincorporated therein or adjacent thereto, and wherein each microscopeslide when disposed in the reaction compartment can be heatedindependently and treated independently of each other microscope slide.17. The apparatus of claim 16 wherein each microscope slide can betreated with a different treatment protocol simultaneously, and/or eachencloseable reaction compartment can be vented by a vapor pressurerelease device to allow release of steam, vapor or pressure therefromwhen the microscope slide is heated, and/or each encloseable reactioncompartment is individually encloseable, and/or comprising amicroprocessor for controlling a component of the apparatus.
 18. Areagent dispensing strip comprising at least one reagent capsule, andsized to be positioned on or inserted into a reagent dispensing stripholder of the apparatus of claim
 16. 19. An in situ antigen recovery andstaining apparatus, comprising: an assembly comprising a plurality ofindividually encloseable reaction compartments wherein the operation ofeach reaction compartment is controllable independently of each otherreaction compartment in the assembly, and wherein each reactioncompartment comprises a slide support element for supporting amicroscope slide, each slide support element having a heating elementincorporated therein or adjacent thereto, and wherein each microscopeslide when disposed in the reaction compartment can be heatedindependently and treated independently of each other microscope slide,wherein the microscope slide can be treated using a reagent dispensingstrip.
 20. The apparatus of claim 19 wherein each microscope slide canbe treated with a different treatment protocol simultaneously, and/oreach encloseable reaction compartment can be vented by a vapor pressurerelease device to allow release of steam, vapor or pressure therefromwhen the microscope slide is heated, and/or each encloseable reactioncompartment is individually encloseable, and/or comprising amicroprocessor for controlling a component of the apparatus.
 21. Areagent dispensing strip comprising at least one reagent capsule, andsized to be positioned on or inserted into a reagent dispensing stripholder of the apparatus of claim
 19. 22. An in situ antigen recovery andstaining apparatus, comprising: an assembly comprising a plurality ofindividually encloseable reaction compartments wherein the operation ofeach reaction compartment is controllable independently of each otherreaction compartment in the assembly, and wherein each reactioncompartment comprises a movable slide support element for supporting amicroscope slide, each movable slide support element having a heatingelement incorporated therein or adjacent thereto, and wherein eachmicroscope slide when supported on the movable slide support element canbe heated independently and treated with a different treatment protocolsimultaneously and independently of each other microscope slide.
 23. Theapparatus of claim 22 wherein each microscope slide can be treated witha different treatment protocol simultaneously, and/or each encloseablereaction compartment can be vented by a vapor pressure release device toallow release of steam, vapor or pressure therefrom when the microscopeslide is heated, and/or each encloseable reaction compartment isindividually encloseable, and/or comprising a microprocessor forcontrolling a component of the apparatus.
 24. A reagent dispensing stripcomprising at least one reagent capsule, and sized to be positioned onor inserted into a reagent dispensing strip holder of the apparatus ofclaim
 22. 25. An in situ antigen recovery and staining apparatus,comprising: an assembly comprising a plurality of individuallyencloseable reaction compartments, and wherein each individuallyencloseable reaction compartment is constructed to contain a singlemicroscope slide, and wherein a reagent on each single microscope slidecontained within the individually encloseable reaction compartment canbe heated to reach a temperature of from about 90° C. to about 100° C.and wherein each single microscope slide can be moved individually. 26.The apparatus of claim 25 further comprising apparatus for automaticallydispensing the reagent onto the microscope slide, and/or wherein eachmicroscope slide can be treated with a different treatment protocolsimultaneously, and/or wherein each encloseable reaction compartment canbe vented by a vapor pressure release device to allow release of steam,pressure or vapor therefrom when the microscope slide is heated, and/orwherein the temperature of the reagent on the microscope slide is 90° C.to 1001C for at least 5 minutes, for at least 10 minutes, and/or for atleast 30 minutes, and/or wherein the addition of additional reagent isnot necessary during heating of the reagent when the reagent on themicroscope slide is heated to 90° C. to 100° C., and/or wherein eachreaction compartment has a volume of up to 15 ml, and/or furthercomprising a microprocessor for controlling a component of theapparatus.
 27. A reagent dispensing strip comprising at least onereagent capsule, and sized to be positioned on or inserted into areagent dispensing strip holder of the apparatus of claim
 25. 28. An insitu antigen recovery and staining apparatus, comprising: an assemblycomprising a plurality of encloseable reaction compartments, and whereineach encloseable reaction compartment is constructed to contain a singlemicroscope slide, and wherein a reagent on each single microscope slidecontained within the encloseable reaction compartment can be heated toreach a temperature of from about 90° C. to about 100° C., and whereineach encloseable reaction compartment can be vented by a vapor pressurerelease device to allow release of steam, pressure or vapor and whereineach single microscope slide can be moved individually.
 29. Theapparatus of claim 28 further comprising apparatus for automaticallydispensing the reagent onto the microscope slide, and/or wherein eachmicroscope slide can be treated with a different treatment protocolsimultaneously, and/or wherein each encloseable reaction compartment canbe vented individually, and/or wherein the vapor pressure release deviceis in the reaction compartment, and/or wherein the temperature of thereagent on the microscope slide is 90° C. to 100° C. for at least 5minutes, for at least 10 minutes, and/or for at least 30 minutes, and/orwherein the addition of additional reagent is not necessary duringheating of the reagent when the reagent on the microscope slide isheated to 90° C. to 100° C., and/or wherein each reaction compartmenthas a volume of up to 15 ml, and/or further comprising a microprocessorfor controlling a component of the apparatus.
 30. A reagent dispensingstrip comprising at least one reagent capsule, and sized to bepositioned on or inserted into a reagent dispensing strip holder of theapparatus of claim
 28. 31. An in situ antigen recovery and stainingapparatus, comprising: an assembly comprising a plurality ofindividually encloseable reaction compartments, and wherein eachencloseable reaction compartment is constructed to contain a singlemicroscope slide, and wherein a reagent on each single microscope slidecontained within the encloseable reaction compartment can be heated toreach a temperature of from about 90° C. to about 100° C., and whereineach encloseable reaction compartment can be vented by a vapor pressurerelease device to release steam, pressure or vapor and wherein theaddition of additional reagent is not necessary during heating of thereagent when the reagent on the microscope slide is heated to 90° C. to100° C.
 32. The apparatus of claim 31 further comprising apparatus forautomatically dispensing the reagent onto the microscope slide, and/orwherein each microscope slide can be treated with a different treatmentprotocol simultaneously, and/or wherein each encloseable reactioncompartment can be vented individually, and/or wherein the vaporpressure release device is in the reaction compartment, and/or whereinthe temperature of the reagent on the microscope slide is 90° C. to 100°C. for at least 5 minutes, for at least 10 minutes, and/or for at least30 minutes, and/or wherein each reaction compartment has a volume of upto 15 ml, and/or further comprising a microprocessor for controlling acomponent of the apparatus.
 33. A reagent dispensing strip comprising atleast one reagent capsule, and sized to be positioned on or insertedinto a reagent dispensing strip holder of the apparatus of claim
 31. 34.An in situ antigen recovery and staining apparatus, comprising: anassembly comprising a plurality of individually encloseable reactioncompartments, and wherein each individually encloseable reactioncompartment is constructed to contain a single microscope slide, andwherein a reagent on each single microscope slide contained within theindividually encloseable reaction compartment can be heated to reach atemperature of from about 90° C. to about 100° C., and wherein eachindividually encloseable reaction compartment can be individually ventedby a vapor pressure release device to release steam, pressure or vapor.35. The apparatus of claim 34 further comprising apparatus forautomatically dispensing the reagent onto the microscope slide, and/orwherein each microscope slide can be treated with a different treatmentprotocol simultaneously, and/or wherein each encloseable reactioncompartment can be vented individually, and/or wherein the vaporpressure release device is in the reaction compartment, and/or whereinthe temperature of the reagent on the microscope slide is 90° C. to 100°C. for at least 5 minutes, for at least 10 minutes, and/or for at least30 minutes, and/or wherein the addition of additional reagent is notnecessary during heating of the reagent when the reagent on themicroscope slide is heated to 90° C. to 100° C., and/or wherein eachreaction compartment has a volume of up to 15 ml, and/or furthercomprising a microprocessor for controlling a component of theapparatus.
 36. A reagent dispensing strip comprising at least onereagent capsule, and sized to be positioned on or inserted into areagent dispensing strip holder of the apparatus of claim
 34. 37. An insitu antigen recovery and staining apparatus, comprising: an assemblycomprising a plurality of encloseable reaction compartments, and whereineach encloseable reaction compartment is constructed to contain a singlemicroscope slide, and wherein each single microscope slide containedwithin the encloseable reaction compartment can be heated to reach atemperature of from about 140° C. to about 160° C., and wherein eachencloseable reaction compartment can be vented by a vapor pressurerelease device to release steam, pressure or vapor.
 38. The apparatus ofclaim 37 further comprising apparatus for automatically dispensing thereagent onto the microscope slide, and/or wherein each microscope slidecan be treated with a different treatment protocol simultaneously,and/or wherein each encloseable reaction compartment can be ventedindividually, and/or wherein the vapor pressure release device is in thereaction compartment, and/or wherein the temperature of the reagent onthe microscope slide is 90° C. to 100° C. for at least 5 minutes, for atleast 10 minutes, and/or for at least 30 minutes, and/or wherein theaddition of additional reagent is not necessary during heating of thereagent when the reagent on the microscope slide is heated to 90° C. to100° C., and/or wherein each reaction compartment has a volume of up to15 ml, and/or further comprising a microprocessor for controlling acomponent of the apparatus.
 39. A reagent dispensing strip comprising atleast one reagent capsule, and sized to be positioned on or insertedinto a reagent dispensing strip holder of the apparatus of claim
 37. 40.An in situ antigen recovery and staining apparatus, comprising: anassembly comprising a plurality of individually encloseable reactioncompartments, and wherein each individually encloseable reactioncompartment is constructed to contain a single microscope slide, andwherein each single microscope slide contained within the individuallyencloseable reaction compartment can be heated to reach a temperature offrom about 140° C. to about 160° C., and wherein each individuallyencloseable reaction compartment can be individually vented by a vaporpressure release device to release steam, pressure or vapor.
 41. Theapparatus of claim 40 further comprising apparatus for automaticallydispensing the reagent onto the microscope slide, and/or wherein eachmicroscope slide can be treated with a different treatment protocolsimultaneously, and/or wherein each encloseable reaction compartment canbe vented individually, and/or wherein the vapor pressure release deviceis in the reaction compartment, and/or wherein the temperature of thereagent on the microscope slide is 90° C. to 100° C. for at least 5minutes, for at least 10 minutes, and/or for at least 30 minutes, and/orwherein the addition of additional reagent is not necessary duringheating of the reagent when the reagent on the microscope slide isheated to 90° C. to 100° C., and/or wherein each reaction compartmenthas a volume of up to 15 ml, and/or further comprising a microprocessorfor controlling a component of the apparatus.
 42. A reagent dispensingstrip comprising at least one reagent capsule, and sized to bepositioned on or inserted into a reagent dispensing strip holder of theapparatus of claim
 40. 43. An in situ antigen recovery and stainingapparatus, comprising: an assembly comprising a plurality of encloseablereaction compartments, and wherein each encloseable reaction compartmentis constructed to contain a single microscope slide, and wherein eachsingle microscope slide contained within the encloseable reactioncompartment can be heated to reach a temperature of from about 140° C.to about 160° C., and wherein each encloseable reaction compartment canbe vented by a vapor pressure release device to release steam, pressureor vapor and wherein the addition of additional reagent is not necessaryduring heating of a reagent disposed on the microscope slide when thereagent on the microscope slide is heated to 90° C. to 100° C.
 44. Theapparatus of claim 43 further comprising apparatus for automaticallydispensing the reagent onto the microscope slide, and/or wherein eachmicroscope slide can be treated with a different treatment protocolsimultaneously, and/or wherein each encloseable reaction compartment canbe vented individually, and/or wherein the vapor pressure release deviceis in the reaction compartment, and/or wherein the temperature of thereagent on the microscope slide is 90° C. to 100° C. for at least 5minutes, for at least 10 minutes, and/or for at least 30 minutes, and/orwherein each reaction compartment has a volume of up to 15 ml, and/orfurther comprising a microprocessor for controlling a component of theapparatus.
 45. A reagent dispensing strip comprising at least onereagent capsule, and sized to be positioned on or inserted into areagent dispensing strip holder of the apparatus of claim
 43. 46. An insitu antigen recovery and staining apparatus, comprising: an assemblycomprising a plurality of individually encloseable reactioncompartments, and wherein each individually encloseable reactioncompartment is constructed to contain a single microscope slide, andwherein each single microscope slide contained within the individuallyencloseable reaction compartment can be heated to reach a temperature offrom about 140° C. to about 160° C.
 47. The apparatus of claim 46further comprising apparatus for automatically dispensing the reagentonto the microscope slide, and/or wherein each microscope slide can betreated with a different treatment protocol simultaneously, and/orwherein each encloseable reaction compartment can be vented by a vaporpressure release device to allow release of steam, pressure or vaportherefrom when the microscope slide is heated, and/or wherein thetemperature of the reagent on the microscope slide is 90° C. to 100° C.for at least 5 minutes, for at least 10 minutes, and/or for at least 30minutes, and/or wherein the addition of additional reagent is notnecessary during heating of the reagent when the reagent on themicroscope slide is heated to 90° C. to 100° C., and/or wherein eachreaction compartment has a volume of up to 15 ml, and/or furthercomprising a microprocessor for controlling a component of theapparatus.
 48. A reagent dispensing strip comprising at least onereagent capsule, and sized to be positioned on or inserted into areagent dispensing strip holder of the apparatus of claim
 46. 49. An insitu antigen recovery and staining apparatus, comprising: a plurality ofslide support elements each for supporting a microscope slide, whereineach slide support element is movable and wherein the treatment of eachmicroscope slide on each slide support element is not dependent upon thetreatment of any other microscope slide on any other slide supportelement.
 50. The apparatus of claim 49 wherein each slide supportelement is independently movable.
 51. An in situ antigen recovery andstaining apparatus, comprising: a plurality of slide support elementseach for supporting a microscope slide, wherein each slide supportelement is independently movable and wherein the treatment of eachmicroscope slide on each slide support element is not dependent upon thetreatment of any other microscope slide on any other slide supportelement.